LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

Despite recent therapeutic advances, the 5-year survival rate for adults with acute myeloid leukemia (AML) is poor and standard-of-care chemotherapy is associated with significant toxicity, highlighting the need for new therapeutic approaches. Recent work from our group and others established that the G protein-coupled estrogen receptor (GPER) is tumor suppressive in melanoma and other solid tumors. We performed a preliminary screen of human cancer cell lines from multiple malignancies and found that LNS8801, a synthetic pharmacologic agonist of GPER currently in early phase clinical trials, promoted apoptosis in human AML cells. Using human AML cell lines and primary cells, we show that LNS8801 inhibits human AML in preclinical in vitro models, while not affecting normal mononuclear cells. Although GPER is broadly expressed in normal and malignant myeloid cells, this cancer-specific LNS8801-induced inhibition appeared to be independent of GPER signaling. LNS8801 induced AML cell death primarily through a caspase-dependent apoptosis pathway. This was independent of secreted classical death receptor ligands, and instead required induction of reactive oxygen species (ROS) and activation of endoplasmic reticulum (ER) stress response pathways including IRE1α. These studies demonstrate a novel activity of LNS8801 in AML cells and show that targeting ER stress with LNS8801 may be a useful therapeutic approach for AML. Significance: Previous work demonstrated that LNS8801 inhibits cancer via GPER activation, especially in solid tumors. Here we show that LNS8801 inhibits AML via GPER-independent mechanisms that include ROS induction and ER activation.


Introduction
Acute myeloid leukemia (AML) is a hematologic malignancy associated with poor differentiation and uncontrolled proliferation of myeloid progenitor cells.
For decades, the first-line therapy for AML has remained the "7 + 3" regimen, Previous reports demonstrated that there are significant GPCR aberrations in AML and that high expression of some GPCRs, including CXCR4, is associated with worse outcomes (17,18). Imipridones, an emerging class of drugs that targets GPCRs, have shown some efficacy in multiple cancer types including AML (19,20).
Recently, the G Protein-coupled estrogen receptor (GPER), a GPCR that functions as a nonclassical surface estrogen receptor, has been shown to have tumor suppressive activity in several cancer types (21)(22)(23)(24)(25). GPER is distinct from classical nuclear estrogen receptors and has been shown to signal through cAMP and calcium in multiple cellular contexts (26)(27)(28)(29). Our group discovered that selective activation of GPER using a synthetic agonist, which does not bind to the classical estrogen receptors, inhibits various cancer types including melanoma and pancreatic ductal adenocarcinoma (30)(31)(32). This GPER-specific agonist, G-1, has recently been shown to inhibit hematologic malignancies, such as T-cell lymphoblastic leukemia and AML, through various mechanisms (33,34). For the first time, we demonstrate that LNS8801, the enantiomerically pure GPER agonist that is currently in early phase clinical trials for melanoma and other advanced malignancies arising in solid organs (31), effectively inhibits AML in vitro and in an in vivo subcutaneous model. This anti-AML activity is mediated by activation of reactive oxygen species (ROS) and ER stress pathways independent of classical GPER signaling.

WST-8 Assay
Cells were seeded at 3 × 10 3 -2 × 10 4 cells per 200 μL media in a clear 96-well plate and were incubated with LNS8801 (provided by Linnaeus Therapeutics), LNS8812 (provided by Linnaeus Therapeutics), or G-1 (Cayman, 10008933) for 72 hours at 37°C with 5% CO 2 . Vehicle (DMSO; Sigma-Aldrich, D8418) concentration was limited to 0.1% for all studies. After incubating cells with appropriate drugs, 10 μL of Cell Counting Kit-8 dye (Dojindo, CK04) were added to each well and absorbance was measured at 450 nm with a Synergy HT plate reader (BioTek). Relative cell number was calculated by the following equation: All experiments were done with five to six replicates per condition.

Cell-cycle Analysis
Cells were plated at a density of 5 × 10 5 cells/mL one day prior to drug treatment. Upon DMSO control or 250 nmol/L LNS8801 treatment, cells were incubated for 20 hours at 37°C with 5% CO 2 . Cells were then washed with dulbecco's phosphate-buffered saline (DPBS) (Corning 21-031-CV) and permeabilized with chilled 70% ethanol. Cells were stained with FxCycle PI/RNase Staining Solution (Invitrogen, F10797) according to the manufacturer's protocol. Flow cytometry on the cells was performed using a BD LSRII flow cytometer, and flow cytometry graphs were analyzed with FlowJo. All conditions were analyzed in triplicate.

CRISPR/Cas9 Knockout and Subcloning
MOLM14 cells expressing Cas9 (MOLM14-Cas9) were a gift from the Carroll lab. MOLM14-Cas9 cells were lentivirally transduced with single-guide RNAs targeting the following sequences: sgGPER3; ATTGAGGTGTTCAACCTGCAC sgGPER5; CTTCTCCAACAGCTGCCTAAAC One week after guide RNA transduction, cells were serially diluted so that there was one cell per well in 96-well plates. Single cells were then expanded and used for functional experiments.

Annexin V/Propidium Iodide Death Assay
AML cell lines were plated at a density of 2.5 × 10 5 cells/mL and were treated with DMSO or drugs for 72 hours at 37°C with 5% CO 2 . To test the functional necessity of ER stress regulators in LNS8801-induced death, cells were plated at a density of 5 × 10 5 cells/mL one day prior to drug treatment. Cells were then treated with drugs for 24 hours. After incubation, cells were harvested and washed with DPBS. Cells were then stained with Alexa fluor 488 conjugated Annexin V antibody and propidium iodide (PI) according to the Cell Death Apoptosis Kit manual (Invitrogen, V13245). Flow cytometry on cells was performed using a BD LSRII flow cytometer, and flow cytometry graphs were analyzed with FlowJo. All conditions were analyzed in triplicate.

High-speed Spectrofluorimetric Ca++ Measurements
For Ca++ measurements, U937 cells were counted and resuspended to a final concentration of 3 × 10 6 cells/mL in complete RPMI. A total of 12 ×

Western Blot Analysis
AML cell lines and AML patient samples were washed once with DPBS and lysed with urea lysis buffer (8 mol/L urea, 50 mmol/L NaCl, 50 mmol/L Tris-

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 8 (GraphPad

Data Availability
The data generated in this study are available upon request from the corresponding author.

LNS8801 Inhibits Human AML in vitro
All prior studies in the literature utilizing a GPER-specific agonist use the synthetic compound G-1 (35). While G-1 is highly selective for GPER over nuclear estrogen receptors, it is a racemic mixture of two enantiomers. As new racemic mixtures are generally not advanced for human use, G-1 was separated into LNS8801 and LNS8812, which do not interconvert ( Fig. 1A and B) (31). All of G-1 s antitumor activity appears to reside in LNS8801, which is the compound currently in human trials.
We tested LNS8801 across a panel of cancer cell lines and found that LNS8801 inhibited growth in multiple cancer types but seemed to promote apoptosis in AML cells ( Supplementary Fig. S1). GPER protein is expressed in AML cell lines and primary AML samples isolated from patients, as well as normal mononuclear cells ( Supplementary Fig. S2A and S2B). To test whether LNS8801 activates classical GPER signaling pathways in AML, we treated primary AML cells with LNS8801 and/or the GPER-specific antagonist G-36 (36) and looked for changes in cAMP-responsive element binding protein (CREB)  phosphorylation, as CREB phosphorylation is a well-established downstream readout of GPER activation (32,37,38). As expected, we found that LNS8801 induced CREB phosphorylation and that the effect was blocked by G-36, suggesting that LNS8801 engages and signals through GPER in primary AML cells ( Supplementary Fig. S2C).
In a panel of commonly used human AML cell lines, we confirmed that the racemic agonist, G-1, and its active enantiomer, LNS8801, reduced relative cell numbers, while the inactive enantiomer LNS8812 failed to do so (Fig. 1C-F).
Consistent with the known heterogeneity in AML drug responses, we noted a range of LNS8801 sensitivity among the four lines (Supplementary Table S1), with U937 being the most sensitive and THP1 being the most resistant. LNS8801 sensitivity did not seem to correlate with mutational status including FLT3 mutation. Similarly, clinical outcomes do not seem to correlate with GPER expression according to BEAT-AML dataset ( Supplementary Fig. S3).
Cell-cycle analysis showed that LNS8801 induced an appreciable decrease in the number of cells in S-phase and a significant increase in sub-G 1 -phase across all AML cell lines (Fig. 1G-J). The loss of cells in S-phase suggests that LNS8801 inhibits proliferation, and the increase in cells in sub-G 1 -phase indicates that LNS8801 compromises cell viability. Together, these data show that LNS8801 effectively inhibits AML in vitro.

LNS8801 Inhibits AML Colony Formation
To test whether the anti-AML activity observed in the established AML lines extended to human primary AML cells isolated directly from patients, we performed colony formation assays using AML patient samples and normal bone marrow mononuclear cells, with or without LNS8801. While LNS8801 significantly reduced the number of colonies in AML patient samples, it did not decrease colony forming capacity in normal mononuclear cells ( Fig. 2A and   B; Supplementary Fig. S4). Colony number and morphology with the normal mononuclear cells were unaffected by LNS8801, which is consistent with the lack of LNS8801 toxicity observed in humans (Supplementary Fig. S4; ref. 31). Characteristics of the primary AML cells are outlined in Supplementary  Table S2.

LNS8801-induced AML Inhibition is Independent of Canonical GPER Signaling
We next tested whether the anti-AML effect of LNS8801 was mediated through GPER and subsequent signaling. To test whether GPER activation leads to AML inhibition, we performed proliferation assays using the endogenous GPER ligand estradiol (E2). Unlike LNS8801, E2 did not inhibit the proliferation of U937 or MOLM14 cells (Fig. 3A and B). Furthermore, we tested the necessity of GPER signaling for LNS8801-induced AML inhibition by using the GPER-specific  Supplementary Fig. S4. One-way ANOVA with multiple comparisons was used to calculate P values for C and D (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).
antagonist G-36 and found that G-36 did not prevent the antiproliferative effect of LNS8801 ( Fig. 3C and D). Finally, we took a genetic approach and partially depleted GPER in MOLM14 using CRISPR-Cas9. After subcloning GPERdepleted cells, we established clonal lines with approximately 70% depletion of the GPER protein ( Supplementary Fig. S5A). Consistent with previous data, the sensitivity of GPER-depleted cells to LNS8801 was similar to the sensitivity of parental cells (Fig. 3E).
GPER canonically activates G s signaling, which increases the intracellular cAMP concentration (39,40). To understand whether GPER activation induces cAMP in AML cells, we measured phosphorylation of CREB, which is a downstream readout of cAMP induction. While we observed an increase in CREB phosphorylation in some AML cell lines and primary AML cells, this did not correlate with LNS8801 sensitivity (Supplementary Fig. S6A and S6B). The CREB phosphorylation in AML cell lines with LNS8801 was less pronounced that what was observed in primary AML. Despite this variability in CREB phosphorylation, LNS8801 inhibited proliferation in all AML cell lines tested.
To further examine whether cAMP signaling contributes to LNS8801-induced inhibition, we treated U937 cells with cAMP analogs and found that none sufficiently inhibited AML cell line growth ( Supplementary Fig. S7A), suggesting that cAMP is not a significant contributor of the LNS8801-induced inhibition in AML.
In other settings, GPER activation leads to increased cytosolic [Ca 2+ ] (40). However, calcium was not affected by LNS8801 nor by E2 in AML, although calcium changes were readily detected after exposure to the thapsigargin, histamine, and ionomycin positive controls ( Supplementary Fig. S7B). This result suggests that LNS8801-induced AML inhibition is also independent of a [Ca 2+ ] flux.

LNS8801 Induces Caspase-dependent Apoptosis in AML
As LNS8801 increased the proportion of cells in the sub-G 1 -phase ( Fig. 1G-J), we hypothesized that LNS8801 induced apoptosis. Consistent with this, LNS8801 treatment increased Annexin V positivity across all cell lines, suggesting that cells died by apoptosis (Fig. 4A). Apoptosis is classically trigged by either the intrinsic or extrinsic apoptotic pathways. The intrinsic apoptotic pathway is regulated by the antiapoptotic Bcl-2 family proteins including Bcl-2, Bcl-xL, and Mcl-1, and the proapoptotic proteins including Bak and Bax. Extreme and extended intracellular stress alters Bcl-2 family protein levels to promote mitochondria pore formation and cytochrome c release, which initiates downstream death signaling pathways (41). In AML, the intrinsic apoptotic pathway is commonly inhibited, but it can sometimes be effectively reengaged in human patients via pharmacologic Bcl-2 inhibition with venetoclax. This led us to question whether the intrinsic apoptotic pathway was responsible for LNS8801-induced cell death. While we did not observe appreciable LNS8801-induced changes in proapoptotic proteins (namely, Bak, Bax, and Bim), we did see consistent depletion of the antiapoptotic protein Mcl-1 across all cell lines ( Supplementary Fig. S8  proteins in AML, such as Bcl-2 and Bcl-xL, were grossly unchanged upon LNS8801 treatment (Supplementary Fig. S8).

Mcl-1 protein is often overexpressed in various cancers and is necessary for
AML survival (42)(43)(44)(45) S9A). We next tested whether restoring Mcl-1 protein expression in the face of LNS8801 exposure prevented LNS8801-induced cell death and found that Mcl-1 overexpression did not prevent LNS8801-induced apoptosis ( Supplementary   Fig. S9B and S9C). Together, these data suggest that LNS8801-induced Mcl-1 loss is not a major determinant of LNS8801-induced cell death in AML.
To further clarify whether LNS8801-induced AML cell death was mediated by the extrinsic or intrinsic apoptosis pathways, we next determined protein levels of cleaved caspases 3, 8, and 9 following treatment with LNS8801. While we observed upregulation of cleaved caspase-8 (extrinsic pathway) and cleaved caspase-3 (downstream effector of both the extrinsic and the intrinsic pathway) in most cell lines, cleaved caspase-9 (intrinsic pathway) was not notably altered by LNS8801 treatment with the exception of the THP1 cell line (Fig. 4B). This indicates that caspases are involved in LNS8801-induced death but that the specific subtype involvement in AML may be context dependent.
Next, to test whether caspase cleavage was a necessary mediator of LNS8801induced death, we used QVD-O-PH (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor). As predicted, the pan-caspase and caspase-8 inhibitor significantly reduced Annexin V positivity in LNS8801-treated U937 cells, while the caspase-9 inhibitor had no effect (Fig. 4C). In MOLM14 cells, the pan-caspase and caspase-8 inhibitor had a more modest effect, indicating that there is heterogeneity in caspase involvement in LNS8801-induced death response (Fig. 4D). These data are consistent with the idea that LNS8801-induced cell death is mediated by activated caspases, including the caspase specific to the extrinsic pathway.
Extrinsic apoptosis is classically triggered by secreted death ligands. Therefore, we questioned whether LNS8801 induces extrinsic cell death by increasing production of these classical death ligands including TNFα, FasL, or TRAIL. We found that there was a robust increase in TNFα expression upon LNS8801 treatment, while FasL and TRAIL were grossly unchanged ( Supplementary  Fig. S10A). To test whether the increased TNFα was responsible for LNS8801induced death, we pretreated AML cells with an anti-TNFR1 blocking antibody and treated cells with LNS8801. While anti-TNFR1 antibody pretreatment blocked death induced by exogenous TNFα, it did not block death induced by LNS8801, suggesting that the LNS8801-induced TNFα increase is not sufficient to kill cells ( Supplementary Fig. S10B).
To test whether different secreted factors activated the extrinsic apoptosis pathway following LNS8801 exposure, we treated AML cells with LNS8801 for 24 hours and collected conditioned media. We then removed LNS8801 from that media via dialysis through a 10,000 molecular weight cut-off (MWCO) membrane, which should retain death ligand proteins. This dialyzed conditioned media did not induce significant cell death, indicating that the "extrinsic" apoptosis observed following LNS8801 is likely initiated by cell intrinsic factors rather than a classical autocrine/paracrine death ligand ( Supplementary Fig. S10C).

LNS8801-induced ROS Production Inhibits AML
Recent reports show that that G-1 induces ROS in other cell settings (33,47).
ROS may cause DNA damage that induces apoptosis, and is a potent inhibitor of AML via various mechanisms (48,49). ROS levels were measured using DCF in U937 and MOLM14 cells after 2 and 4 hours of treatment with LNS8801.
LNS8801 significantly increased the levels of ROS in both cell lines, while the inactive enantiomer LNS8812 did not induce ROS (Fig. 5A-D; Supplementary   Fig. S11). We then tested whether LNS8801-induced ROS upregulation is necessary for AML death. The ROS scavenger NAC blunted LNS8801-induced ROS production as well as the associated cell death (Fig. 5E-H). While we tried to test whether higher concentration of NAC further reversed the effect of LNS8801, we were unable to do so as NAC itself was toxic at higher concentrations, which is consistent with observations from other groups (50,51).
Together, these data show that LNS8801 leads to increased production of ROS, which induces apoptosis in AML cells.

LNS8801 Induces Apoptosis Through the ER Stress Pathway
ROS-induced cell death is often associated with ER stress (52)(53)(54), and G-1 induces ER stress pathway in some solid tumor models (24,55). Therefore, we questioned whether LNS8801 induced apoptosis in AML via the ER stress pathway. The main ER stress sensors that govern apoptosis are IRE1α and PERK (56). Upon activation of IRE1α and PERK, downstream effectors including XBP1 and CHOP are upregulated, which then drive apoptosis (57,58). To test whether LNS8801 induced activation of these ER stress responses in AML, we determined levels of ER stress proteins and noted increases in both XBP1 and CHOP ( Fig. 6A and B). Unlike LNS8801, E2 did not induce any noticeable increase in XBP1 or CHOP expression ( Supplementary Fig. S12A and S12B).
To test whether these ER stress responses are a necessary for LNS8801induced cell death, we used ER stress inhibitors in combination with LNS8801.

LNS8801 Inhibits AML in Subcutaneous In Vivo Model but not Systemic Model
We next tested whether LNS8801 inhibited AML in preclinical in vivo models. Initially, NSG mice were injected with luciferase-expressing MOLM14 cells (MOLM14-Luc) to test the efficacy of LNS8801 in a clinically relevant setting. Mice harboring MOLM14-Luc xenografts were treated once daily with orally delivered LNS8801 (100 mg/kg). This dosage is approximately 20-100 times higher than what has been used in other preclinical models (30,32,59). To our surprise, LNS8801 did not inhibit AML in the systemic model (Fig. 7A). This observation was consistent in another AML cell line, MV4-11, that is sensitive to LNS8801 in vitro (Supplementary Fig. S14A and S14B). To understand whether LNS8801 inhibits AML in different in vivo contexts, we engrafted MOLM14 cells to NSG mice subcutaneously. Xenografted mice were treated once daily with orally delivered LNS8801 (mice were treated with 10 mg/kg LNS8801, which is approximately 2-10 times the dosage of what has been used in other preclinical models (30,32,59)) once tumors reached 150-250 mm 3 in size. LNS8801 significantly inhibited MOLM14 tumor growth and extended median survival from 3 to 9 days ( Fig. 7B and C).

Discussion
Here, we show that LNS8801 promotes apoptosis in AML through upregulation of ROS, activation of ER stress, and downstream initiation of caspase-dependent apoptosis pathways. Consistent with the lack of toxicity observed in ongoing clinical studies, LNS8801 did not affect normal mononuclear cells.
While these data suggest that LNS8801 inhibits AML through caspase activation, the death appears independent of classical secreted death ligands and changes in Bcl-2 family proteins. Rather, AML death is dependent on ROS and ER stress responses. GPER signaling canonically induces an increase in both intracellular cAMP and calcium. However, our data suggest that the effects of LNS8801 on apoptosis in AML do not depend on these classical G-protein signaling pathways.
As GPCR signaling can often vary depending on cell context (49), it is possible that LNS8801 inhibits AML via nonclassical GPER signaling. Upon activation, GPCRs release the βγ G-protein subunit that has known signaling properties. It has been shown that the βγ subunit coupled to GPER increases MAPK signaling in different cellular contexts (50), indicating that there are multiple pathways outside of the canonical GPER signaling pathway that may mediate the effect of LNS8801. This is consistent with recent data from other groups demonstrating that G-1 inhibits some AML lines through a GPER-p38 signaling axis (34). In addition, GPCR kinases may play a role, as it is becoming evident that they have signaling capacity independent of GPCRs that could potentially explain some of nonclassical GPER signaling (60). or vehicle for MFI DCF measurements. Cells were pretreated with 4 mmol/L NAC or vehicle for 1 hour, followed by 24-hour treatment of 250 nmol/L LNS8801 or vehicle for annexin V+ measurements. Statistical analysis was done via one-way ANOVA with multiple comparisons was used for B, D, and E-H (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

AACRJournals.org
We attempted to deplete GPER protein in AML cells using both short hairpin RNA and CRISPR-Cas9 approaches. While we observed reductions in GPER protein, these were not complete nor durable, as GPER protein quickly returned to baseline levels, making definitive functional experiments challenging. It is likely that acute GPER loss is not tolerated in AML cell lines. To circumvent technical limitation, we used the GPER-specific antagonist G-36, which was unable to block LNS8801 induced AML cell death. In addition, reduction in GPER protein using genetic approaches did not alter sensitivity to LNS8801. This is consistent with findings in uveal melanoma, where LNS8801 also induces cell death in vitro independent of GPER (59). More complete and durable GPER depletion will be required to understand the effect of GPER-dependent signaling in AML and definitively exclude a role for GPER in LNS8801-induced AML death. Nonetheless, based on the data presented here, it appears that LNS8801 promotes AML cell death independent of classical GPER signaling. It should be noted that while most of the AML cell lines treated with LNS8801 upregulated cleaved caspase-8, there was only a modest increase in cleaved caspase-8 and a more notable increase in cleaved caspase-9 in the (relatively) LNS8801-resistant line THP1. Also, while the caspase-8 inhibitor effectively blunted LNS8801-induced apoptosis in U937 cells, its effect was less pronounced in MOLM14 cells. This suggests that LNS8801 can promote apoptosis in AML cells through multiple mechanisms that are not limited to caspase-8 cleavage. Consistent with this, G-1 induced pyroptosis in addition to AACRJournals.org Cancer Res Commun; 3(8) August 2023 apoptosis in AML cell lines OCI-AML2 and KG1a (34). These alternative mechanisms warrant further investigation, as AML is a highly heterogeneous disease and the mechanisms described in this work may not capture all of the AML complexity.
Ren and colleagues demonstrated that depletion of Mcl-1 is a major mediator for G-1/venetoclax combination-induced cell death in AML through GPER-p38 signaling, but did not investigate whether Mcl-1 depletion is necessary for G-1-induced death (34). To test whether Mcl-1 depletion plays a role in LNS8801-induced death, we overexpressed Mcl-1 in AML cells so that Mcl-1 protein is maintained when treated with LNS8801 and found that the rescue of Mcl-1 did not prevent cell death. Taken together, these data suggest that LNS8801 may be therapeutically useful in the context of venetoclax-resistant AML, because one of the major mechanisms of venetoclax resistance is driven by Mcl-1 upregulation.
While LNS8801 clearly inhibits AML in in vitro contexts, the in vivo efficacy is unclear, as LNS8801 inhibited AML only in the subcutaneous model and not the systemic model. Likely explanations for this discrepancy include pharmacokinetic differences between tissues and compensatory mechanisms provided by the bone marrow niche. LNS8801 is highly fat soluble, mostly protein bound, and metabolized fairly quickly in mice, so it is possible that AML cells in systemic models treated once daily with LNS8801 are not sufficiently exposed to bioavailable compound in the blood (61). Furthermore, because the subcutaneous tissue is high in fat (62), LNS8801 may concentrate in the subcutaneous space and thereby contribute to increased AML sensitivity to LNS8801 in the subcutaneous versus the circulating in vivo models. It is well known that the bone marrow niche provides compensatory survival and proliferation advantages to leukemic cells through cytokines and endothelial cell support, indicating that AML cells in the systemic model may be rescued by tumor microenvironmental factors (63,64).
LNS8801 is well tolerated in humans and appears efficacious in some patients with solid tumors (65); however, the efficacy of LNS8801 in people with hematologic malignancies has not yet been tested. While LNS8801 did not inhibit AML in the systemic model, it may still be effective in patients because the half-life is significantly longer in humans; however, a higher concentration of LNS8801 may be required for AML compared with solid tumors. Furthermore, combination therapy with LNS8801 may prove effective to overcome potential compensatory mechanisms associated with the bone marrow niche. Importantly, of the five primary AML cell samples used in our study, all were sensitive to LNS8801, including two samples (5575 and 5868) isolated from a secondary leukemic and a patient with relapsed leukemic. These types of leukemia are mostly resistant to conventional chemotherapy. If LNS8801 proves to be an effective agent in patients with AML, future tri-als testing the efficacy of LNS8801 in patients with refractory AML may be warranted.